Membrane proteins are crucial in a wide range of biological functions including respiration, signal transduction and mediation of molecular traffic in and out of cells and organelles. In particular, membrane proteins play an important role in drug uptake and distribution and can therefore profoundly affect drug therapy and resistance to treatment by multiple drugs. By way of illustration, and without limitation, MDR1 P-glycoprotein (P-gp) is an ATP-driven low-specificity efflux pump playing a paramount role in the clearance of xenotoxins. This member of the ATPase Binding Cassette (ABC) transporters is a natural barrier against hydrophobic cytotoxic compounds, as well as natural products, cyclic and linear peptides. On the one hand, its overexpression in tumour cells, impairing targeted drug delivery, is a major pitfall for actual chemotherapies. On the other hand, recent studies have shown that gene therapies promoting the expression of MDR1 specifically in pluripotent hematopoietic stem cells could protect them from chemotherapeutics.
Despite their importance, membrane proteins and ligand-bound complexes thereof are notoriously difficult to study. X-ray crystallography is the standard technique for confirming the presence and position of ligands in the binding sites of membrane proteins. However, the conformational flexibility of a membrane protein or a membrane protein-ligand complex is restricted within a crystal lattice and may distort the structural and/or ligand-binding properties of a protein. Furthermore, difficulty is often encountered with the crystallisation of membrane proteins and membrane protein-ligand complexes.
Other methods for characterising membrane proteins and membrane protein-ligand complexes are also known, such as gel electrophoresis, analytical ultracentrifugation and X-ray scattering. However, these methods are low-resolution methods and usually require large quantities of protein. In addition, data from these methods do not normally reveal ligand binding to membrane proteins, or allow observation on post-translational modifications. In particular, the methods do not provide detailed information regarding drug binding to membrane proteins. Drug binding to proteins is often measured using indirect methods such as fluorescence or calorimetry. However, these methods do not provide structural or conformational information regarding the bound protein complex.
Until recently, mass spectrometry had not been considered suitable for detecting intact membrane proteins. This was primarily because of the insolubility of membrane proteins in buffers compatible with electrospray, as well as the ready dissociation of subunit interactions, both between transmembrane subunits and between transmembrane and cytoplasmic subunits, as a result of the transition of the protein into the gas phase environment of a mass spectrometer. However, the detection of a membrane protein, a membrane protein cooperatively bound to adenosine triphosphate (ATP) and a membrane protein bound with a lipid by electrospray ionisation-mass spectrometry has now been demonstrated (see Barrera et al, Science 2008, 321, 243-246; Barrera et al, Nat. Methods 2009, 6, 585-587; and Wang et al, J. Am. Chem. Soc. 2010, 132, 15468-15470). Despite these advances, the use of mass spectrometry to characterise the binding of membrane proteins to therapeutic agents such as drugs has not yet been demonstrated.